Diagnostic procedure



United States Patent 3,201,202 DIAGNOSTIC PRGCEDURE Ronald L. .Searey, Monterey Park, and Lois M. Bergquist,

North Hollywood, Calif., assignors to Hyland Laboratories, Los Angeles, Calif. No Drawing. Filed Mar. 4, 1963, Ser. No. 262,316

2 Claims. (6!. 23-230) This invention is concerned with a novel method for the micro-quantitation of proteins contained in biological fluids, such as cere'brospinal fluid, urine and pleural elfusions. More particularly this invention is concerned with the quantitation of albumin and globulin, both separately and as total protein, through the use of micro-amounts of a test specimen, such as blood serum.

Although the biuret procedure is the classic methodology for the quantitation of proteins contained in biological fluids, it does have certain disadvantages. For example, in the routine laboratory quantitation of total blood protein, amounts of blood serum on the order of 0.5 ml. are required for biuret determinations. In order to ob tain these quantities of blood serum, it is necessary that the patients blood be withdrawn by venepuncture. If smaller amounts of blood would sui'iice, finger-prick blood could be utilized. Also, when biuret reagent is added to a protein-containing specimen, about minutes is required for the full development of the color attributable to the biuret-protein combination. This 'biuret-protein color is light-sensitive and must be read colorimetrically at its peak intensity, before it begins to fade.

It is the object of this invention to provide a simplified colorimetric methodology for the quantitation of proteins in biological fluids.

It is further an object to provide a test procedure for determining proteins in an amount of a biological fluid specimen on the order of as little as 0.001 ml.

It is yet another object to provide, for the quantitation of proteins in biological fluids, a colorimetric procedure which employs light-stable color reagents.

Other objects and advantageous features will become apparent from the following detailed description of the invention.

The instant inventive concept is based upon a technique which employs the dye substance, Ponceau S, as a color reactant for combination with proteins. Although the invention will, hereafter be characterized in relation to blood serum as a protein-containing test specimen, it will be understood that the scope of the invention is not to be limited to such a test specimen, but it is to encompass all protein-containing biological fluids.

In the practice of the invention, a small aliquot of a blood serum specimen, obtained, for example, by finger prick, is applied to a surface which is adsorbent for the serum. The adsorbent surface may comprise any material which is capable of absorbing the serum, permitting the proteins contained in the serum to react with the Ponceau S dye without leaching of the protein into the dye solution, and which is further capable of permitting the protein-bound Ponceau S to besubsequently eluted from the material. Cellulose acetate paper has been found to possess the requisite characteristics.

A small segment of cellulose acetate paper to which an aliquot of blood serum has been applied is placed on the surface of a quantity of Ponceau S solution. The dye is permitted to react with and become chemically bound to 3-,Z0l,202 Patented Aug". 1?, 1905 ice the serum proteins. Unbound dye is then rinsed from the cellulose actate paper.

The protein-bound dye is then eluted from the cellulose acetate papers under alkaline conditions, and Ponceau S is quantitated colorimetrically, either at 560 millimicrons using a spectrophotometer or using a green filter (#54 Klett). The requisite alkalinity can be provided, for example, by an aqueous alkali metal hydroxide solution. Ponceau S in alkaline solution is purple. The amount of Ponceau S extracted from the cellulose acetate paper is directly proportional to the seriurn protein concentration. Since Ponceau S is exceedingly stable in the presence of light, the alkaline eluate can be stored for an indefinite period of time before the colorimetry need be performed.

The reaction between Ponceau S and serum proteins occurs very quickly. Approximately five minutes is required for the completion of this reaction. This period of time will permit the absorption of serum protein concentrations of from at least 3 to 16 gm. percent, covering the entire normal range and a majority of the abnormal serum protein concentrations. Table 1 demonstrates how rapidly the protein in serum applied to cellulose acetate papers can be stained with Ponceau S.

TABLE 1.STAINING SERUM PROTEIN WITH Each result obtained from 0.004 ml. of pooled serum applied to cellulose acetate.

The rapidity of elution of the dye from the Ponceau 5- protein complex under alkaline conditions is shown in Table 2..

, TABLE 2 Extraction time (min):

Optical density at 560 mu 1 Performed in duplicate. Each result obtained from 0.004 ml. of pooled serum applied to cellulose acetate.

The use of Poneeau S in accordance with this invention may also be employed to measure serum albumin and globulin concentrations separately. In this procedure he total protein is first determined by the above methodology. To a second aliquot of serum is then added a substance, such as alcoholic trichloroacetic acid, which causes serum globulins to precipitate and which permits serum albumins to remain in solution. After centrifuga tion, the albumin-containing supernatant is applied to cellulose acetate paper, stained with Ponceau S and quantitated in the .same manner for total protein, and the globulin is obtained by arithmetic difference.

s not see 8b The invention will be more clearl illustrated '0 refer- J ence to the following detailed examples.

Example 1 Serum total protein is determined as follows:

(1) A 0.004 ml. aliquot of serum is applied to a small segment of cellulose acetate paper.

(2) The cellulose acetate paper is then placed on the surface of several drops of 0.2% aqueous solution of Ponceau S and is permitted to remain in contact with the Ponoeau S for five minutes.

(3) The cellulose acetate papers are then dipped in distilled water to rinse off the unbound dye.

(4) The cellulose acetate papers are then placed in cuvettes which contain 6.0 ml. of 0.1 N sodium hydroxide and are permitted to remain in contact with the sodium hydroxide for five minutes.

(5) The amount of dye extracted is directly proportional to the serum protein concentration and is quantitated by reading absorbence (O.D.) at 560 millimicrons wave length on a spectrophotometer. For such quantitation, the conventional photometric procedures, employing a known control and a reagent blank, are utilized.

Example 2 The Ponceau S techniques of this invention may be used to measure serum albumin and globulin concentrations in the following manner:

(1) Total protein levels are measured as described above after applying a 0.004 ml. aliquot of a serum specimen to cellulose acetate paper.

(2) A 0.1 ml. aliquot of the same serum specimen is added to 0.9 ml. of one percent trichloroacetic acid, in 96% ethanol thereby precipitating the serum globulins.

(3) After centrifugation, 0.01 ml. of the supernatant fiuid is applied to cellulose acetate paper stained with Ponceau S, and the albumin is quantitated in the same manner for total protein in Example 1.

(4) The globulin contained in the specimen is obtained by subtracting the measured quantity of albumin protein from the measured total protein.

Example 3 Tables 3 and 4, which follow, demonstrate the close correlation between the procedures of this invention and the conventional procedures for the quantitation of blood serum albumin and globulin. For such quantitation, the globulins in a serum sample were precipitated by admixing one part of serum and nine parts of 1% alcoholic trichloroacetic acid. After centrifugation of the admixture, microliters of the albumin-containing supernatant were transferred to cellulose acetate segments. These segments were then transferred to the surface of one ml. of a 0.2% aqueous solution of Ponceau S and were permitted to remain in contact with the dye for five minutes. The unbound dye was rinsed from the segments. Tht protein-bound dye was then eluted from the segments by placing the segments in cuvettes which contained 6.0 ml. of 0.1 N sodium hydroxide. After the segments had been in contact with the alkaline solution for five minutes, the eluted dye was quantitated by reading absorbence (O.D.)' at 560 millimicrons wave length on a spectrophotometer, as in Example 1. Optical density readings were converted into gm. percent albumin by use of a serum of known protein concentration. Measurements made in this manner were compared to values obtained by the conventional salt fractionation technique of Wolfson and co-workers (Am. J. Clin. Path. 18, 723; 1948), followed by the conventional biuret color development determinations (J. Biol. Chem, 177, 751-756; 1949). A series of comparisons of albumin values obtained by these two methods are shown in Table 1. These values represent single determinations on each of 20 different serum samples and they show an excellent correlation between the two methods.

TABLE 3 .MEASUREME IT OF SERUM ALBUlvllN BY TWO TECHNIQUES [Albumin concentrations (gm. pcrccnt)] Ponceau S Biuret Ponccau S Biurct TABLE 4.-MEASUREMENT OF SERUM GLOBULIN BY TWO TECHNIQUES [Globulin concentration (gm. Pcrcentfl Ponceeu S Biuret Ionceau S Biuret 2. 4 2. 6 2. 9 3. 2 4.1 4. 2 2. 9 3.1 3.1 3. 4 3. 4 3. 7 3.1 3. 4 2. 5 2. 8 8. 2 3. 0 3. l) 4. 0 3. 4 3. 2 4. 2 4. 0 3. 5 3. 4 3. 2 3.2 2.8 3.0 3.1 3.1 3. 8 4. 0 2. E) 2. 6 3.9 4. 0 3. 2 3. a

It will be readily apparent to those skilled in the art that a wide variety of modifications may be made without departing from the spirit and scope of the invention.

What is claimed is:

1. in a method for the quantitation of total proteins in a biological fluid specimen, the steps of:

(a) Adsorbing a measured aliquot of the specimen on an adsorbent material (1) which is capable of accepting the specimen so as to permit the proteins to react with a solution of the dye, Ponceau S, to form a protein-dye complex without leaching of the proteins into t.e dye solution, and (2) which permits the protein-bound dye to be eluted from the adsorbent material under alkaline conditions;

(b) Placing the specimencontaining adsorbent material on the surface of a solution of Ponceau S, to thereby cause all of the protein in the specimen to react with Ponceau S.

(c) Rinsing from the adsorbent material all of that amount of the Ponceau S that has not reacted with the protein.

(d) Eluting the protein-bound Ponceau S from the adsorbent material under alkaline conditions.

(e) Colorimetrically quantitating the eluted Ponceau S and converting the optical density readings thus obtained into total protein concentration.

2. In a method for the quantitation of albumin in a biological fluid specimen, the steps of:

(a) Determining total protein on an aliquot of the specimen by the method or" claim 1. a

(b) Adding a second measured aliquot of the same specimen to a known volume of trichloroacetic acid to thereby precipitate the globulins in the serum.

5 6 (c) Adsorbing a measured amount of the albumin- (g) Colorimetrically quantitating the eluted Ponceau containing, glflbulill-free Supernatant from p S and converting the optical density readings thus on a material which is capable of accepting the obtained into albumin concentration. supernatant so as to permit the albumin to react with a solution of the dye, Ponceau S, to form an 5 References Ci by the Examiner albumin-dye complex Without leaching of the a1bu min into the dye solution, and (2) which permits UNITED STATES PATENTS the albumin-bound dye to be eluted from the ad- 2 Gomberg sorbent material under alkaline conditions. 3O98719 7/63 Skeggs 23*253 (d) Placing the albumin-containing adsorbent material 10 i on the surface of a solution of Ponceau S, to thereby OTHER REFERENCES 53 1 2 2 the Protem m the speclmen to react with Allpoi't, Colorimetric Analysis, published by Chap- (e) Rinsing from the adsorbent material all of that man and Hall 1947 (Pages 262-263 relied alrlroglribtutrilffithe Ponceau S that has not reacted with 15 MORRIS o- WOLK, Primary Examiner (f) Eluting the albumin-bound Ponceau S from the ad- JAMES H. TAYMAN, IR Examiner.

sorbent material under alkaline conditions. 

1. IN A METHOD FOR THE QUANTITATION OF TOTAL PROTEINS IN A BIOLOGICAL FLUID SPECIMEN, THE STEPS OF: (A) ADSORBING A MEASURED ALIQUOT OF THE SPECIMEN ON AN ADSORBENT MATERIAL (1) WHICH IS CAPABLE OF ACCEPTING THE SPECIMEN SO AS TO PERMIT THE PROTEINS TO REACT WITH A SOLUTION OF THE DYE, PONCEAU S, TO FORM A PROTEIN-DYE COMPLEX WITHOUT LEACHING OF THE PROTEINS INTO THE DYE SOLUTION, AND (2) WHICH PERMITS THE PROTEIN-BOUND DYE TO BE ELUTED FROM THE ADSORBENT MATERIAL UNDER ALKALINE CONDITIONS; (B) PLACING THE SPECIMEN-CONTAINING ADSORBENT MATERIAL ON THE SURFACE OF A SOLUTION OF PONCEAU S, TO THEREBY CAUSE ALL OF THE PROTEIN IN THE SPECIMEN TO REACT WITH PONCEAU S. (C) RINSING FROM THE ADSORBENT MATERIAL ALL OF THAT AMOUNT OF THE PONCEAU S THAT HAS NOT REACTED WITH THE PROTEIN. (D) ELUTING THE PROTEIN-BOUND PONCEAU S FROM THE ADSORBENT MATERIAL UNDER ALKALINE CONDITIONS. (E) COLORIMETRICALLY QUANTITATING THE ELUTED PONCEAU S AND CONVERTING THE OPTICAL DENSITY READINGS THUS OBTAINED INTO TOTAL PROTEIN CONCENTRATION. 